Hexahydroindane dicarboxylic acid derivatives

ABSTRACT

4 Alpha -(-Carboxyethyl)-5-oxo-(1 Alpha -hydrogen- or 1 Alpha hydroxyl)-7 Alpha Beta -methyl-3 Alpha Mu -hexahydroindanecarboxylic acids or lower alkanoic acids, which can be prepared by one-step fermentative degradation of the bile acids or their homologues, are useful as cholesterol lowering agents.

United States Patent Hayakawa et al.

[ 1 July 4, 1972 [54] HEXAHY DROINDANE DICARBOXYLIC ACID DERIVATIVES [72] Inventors: Shohei Hayakawa, Amagasaki; Yoshiko Kanematsu, Kyoto; Takashi Fujiwara,

Amagasaki, all of Japan [73] Assignee: Shionogi 8; Co., Ltd., Osaka, Japan [22] Filed: Sept. 25, 1970 [21] Appl.No.: 75,728

Related US. Application Data [60] Division of Ser. No. 771,994, Oct. 29, 1968, Pat. No. 3,560,558, which is a continuation-in-part of Ser. No. 478,735, Aug. 10, 1965, abandoned.

.[52] US. Cl. ..260/514 R [51] Int. Cl ..C07c 61/32 I [58] Field of Search ..260/5l4 [5 6] References Cited UNITED STATES PATENTS 3,616,228 10/1971 Schubert et al ..l95/5l Primary Examiner-Lorraine A. Weinberger Assistant Examiner-Robert Gerstl Attorney-E. F. Wenderoth et al.

[57] ABSTRACT 3 Claims, No Drawings HEXAHYDROINDANE DICARBOXYLIC ACID DERIVATIVES This application is a divisional application of copending U.S. Pat. application, Ser. No. 711,994 filed Oct. 29, 1968, now US. Pat. No. 3,560,558, which is a continuation-in-part application of copending U.S. Pat. application, Ser. No. 478,735, filed Aug. 10, 1965 now abandoned.

This invention relates to novel hexahydroindane dicarboxylic acid derivatives. More particularly, it relates to novel useful compounds represented by the general formula:

X-C OOH car-om wherein X means absence or presence of a grouping selected from Cl-l -CH(CH and --CH(CH )-(CH in which n is an integer from 1 to 4 inclusive; and R is a hydrogen atom or a hydroxyl group. Compounds I are useful as cholesterol metabolism controlling agents for treatment of hypercholesterolemia in human and other warm-blooded animals.

The object of the present invention is to provide novel useful compounds. Another object is to provide novel cholesterol metabolism controlling agents having no significant side effects.

Compounds 1 can be prepared by a process which comprises cultivation of Corynebacterium (Arthrobacter) simplex in a culture medium containing one or more following steroidal substances of the formula:

1; x coon wherein X and R have the same significances as defined above; Y means a hydrogen atom or hydroxyl group; and Z is selected from the group consisting of:

wherein Y is a hydrogen atom or hydroxyl group; W is an a-or fl-hydroxyl group or 0x0 group; and the broken line represents the absence or presence of a double bond, as main carbon sources.

Among the starting compounds which fall in the above designation, the following are smoothly consumed and converted by the Corynebacterium according to the hereindescribed process: 301, 7a, lZa-trihydroxy-SB-androstanel 7fl-carboxylic acid, 3-oxo-SB-androstanel 7B-carboxylic acid, 3-oxo-androst-4-ene-l7B-carboxylic acid, Zia-hydroxyandrost-5-ene-l7fi-carboxyllc acid, 3-oxo-androsta-l,4-

diene-l7B-carboxylic acid, 3-oxo-l7a-hydroxy-SB-androstane-l7/3-carboxylic acid, 3-oxo-l7a-hydroxy-5a-androstane-l7B-carboxylic acid, 3-oxo-l7a-hydroxyandrost-4- ene- 1 7fi-carboxylic acid, 3-oxo-l 7a-hydroxyandrostl ,4- diene-17B-carboxylic acid, 30:, l7a-dihydroxyandrastane-l7fi -carboxylic acid, 33, l7a-dihydroxyandrostane-l7,8-carboxylic acid, 33, l7a-dihydroxyandrost-5-ene-l7B-carboxylic acid, 3 :o o -23f:ng 5@cholan;22-cjc acid, 3-oxo-23-norchol-4-en- 22-oic acid, 3B-hydroxy-23-norchol-5-en-22-oic acid, 3-oxo- 23-norchola-1,4-dien-22-oic acid, 3-oxo-23-norchol5-en-22- oic acid, 3-oxo-l7a-hydroxy-23-norchol-4-en-22-oic acid, 3- oxo-l7a-hydroxy23-norchol-5-en-22-oic acid, BB-hydroxyl7mhydroxy-23-norchol-5-en-22-oic acid, 3-oxo-l7crhydroxy- ;n9r .h9, a:lfia fl- 'l -h1d9? l1?:; norchol-5-en-22-oic acid flithoitholid acid, deoxycholic acid,

cholic acid, 3-oxo-chol-4-enic acid, SB-hydroxychol-S-enic acid, 3-oxo-7a, l2a-dihydroxy-chol-4-enic acid, 3-oxo-chola- 1,4-dienic acid, 3-oxo-l2a-hydroxychola-4,6-dienic acid, 3-

oxo-l7a-hydroxychol-4-enic acid, 33, l7a-dihydroxychol-5- enic acid, 3-oxo-7a, 12a, l7a-trihydroxychol-4-enic acid, 3-

oxo-l7a-hydroxychola-1,4-dienic acid, 3-oxo-l2a-hydroxychola-4,6-dienic acid, l7a-hydroxy-lithocholic acid.

Other compounds which also fall in the same designation are not smoothly consumed and converted solely in the reaction. However, such compounds can also be utilized when the enzyme system in the used microbe is previously or simultaneously adapted or induced by contact with the above-mentioned easily utilizable substrates. For such procedure, the following compounds may be illustrated as the starting materials: 3a-hydroxy-5B-androstane-l7/3-carboxylic acid, 3a, 12adihydroxy-Sfi-androstane-l7B-carboxylic acid, 3a, 7adihydroxy-SB-androstane-l7/3-carboxylic acid, 3-oxo-5a-androstane-l 7/3-carboxylic acid, 3B-hydroxyandrost-5-ene-l 7B- carboxylic acid, 3-oxo-12a-hydroxyandrosta-4,6-diene-l 7B- carboxylic acid, Ba-hydroXy-SBpregnan-Zl-Oic acid, 3-oxo-5a -pregnan-21-oic acid, 3-oxopregn-4-en-2l-oic-aeid, 3B- hydroxypregn-S-en-Z l -oic acid, 3-oxopregn-l ,4-dien2 1 -oic acid, 3a-hydroxy-23-nor-SB-cholan-Z2-oic acid, 30:, 12a dihydroxy-23-nor-5B-cholan-22-oic acid, 30:, 7a-dihydroxy- 23-nor-5/3-cholan-22-oic acid, 30:, 7a, l2a-trihydroxy-23-nor- 5B-cholan-22-oic acid, 3-oxo-12a-hydroxy-Z3-nor-5B-cholan- 22-oic acid, 3-oxo-23-nor-5a-cholan-22-oic acid, 30:, l2adihydroxy-24-nor-5B-cholan-23-oic acid, 30:, 12atrihydroxy-24-nor-5B-cholan-23-oic acid, 3-oxo-24-nor-5acholan-23-oic acid, 3-oxo-24-norchol'4-en-23-oic acid, 33- hydroxy-24-norchol-5-en-23-oic acid, 3-oxochola-l ,4-dien-24 -oic acid, chenodeoxycholic acid, 3-oxo-5B-cholanic acid, 3- oxo-l2a-hydroxy-5fi-cholanic acid, 3-oxo-5a-cholanic acid, 3-oxo-l2a-hydroxy-5a-cholanic acid, 3-oxo-5/3-chol-l-enic acid, 3-oxo-12ahydroxy-5B-chol-l-enic acid, 3-oxo-5a-choll-enic acid, 3-oxo-l2a-hydroxychol-4-enic acid, Zia-hydroxychol-S-enic acid, 3a, IZa-dihydroxychol-S-enic acid, 33, l2a-dihydroxy-chol-5-enic acid, 3oxo-l2a-hydroxychola- 1,4dienic acid, 30:, 7a, lZa-trihydroxy-SB-cholane-24-carboxylic acid, 3-oxo-5B-cholane-24-carboxylic acid, 3-oxochol-4-ene-24-carboxylic acid, 3B-hydroxychol-5-ene-24- carboxylic acid, 3a-hydroxy-27-nor-5B-cholestan-26-oic acid, 3a, 7a, lZa-trihydroxy-Z7-nor-5l3-cholestan-26-oic acid, 3- oxo-27-nor-5/3-cholestan-26-oic acid, 3-oxo-27-norcholest-5' en-26-oic acid, 3a-hydroxy-SB-cholestan-Zfi-oic acid, 30:, 12a -d.ihydroxy-5B26-oic acid, 30:, 7a-dihydroxy-5B- cholestan-26-oic acid, 3a, 7a, lZa-trihydroxy-SB-cholestan- 26-oic acid, 3-oxo-5a-cholestan-26-oic acid, 3B-hydroxycholest-5-en-26-oic acid, 33, l7a-dihydroxy-23-norchol-5-en- 22-oic acid, 3-oxo-l7a-hydroxy-chol-4-ene-24-carboxylic acid, 3-oxo-l7a-hydroxy-5B-cholane-24-carboxylic acid.

They are used in the reaction not only in the free acid form but also in the corresponding water-soluble salt form such as sodium salt or potassium salt. The conjugated compounds such as glycoand tauro-cholic acid, -deoxycholic acid, lithocholic acid, etc. or a mixture thereof, especially crude preparations of ox bile extract, etc., can also be used effectively.

The reaction can be carried out by the conventional aerobic cultivation method except for using Corynebacterium simplex and the above-mentioned steroidal substances solely or in combination as the carbon source. However, in many cases, the addition of conventional carbon sources such as glucose decreases the yield of the products. The reaction can be car ried out at a temperature between about 25 C. and about 35 C, a pH between about 6.5 and about 8.0, with agitation and aeration. The culture medium should contain nitrogen source such as peptone, urea, ammonium sulfate or the like, and several inorganic salts found generally to be effective to promote the growth of microorganisms. Maximum yield of the products is generally realized in from about 1 to days. The optimal reaction period can be preferably determined by checking with chromatographic techniques, especially with thin-layer chromatography. The yield of the product and optimal reaction period varies with kind of starting compound used or method of induction or adaptation. It is generally noted that lower molecular weight starting compounds require shorter reaction periods and afford higher yields.

Specific examples of products thereby obtained are lB-carboxy-5-oxo-7aB-methyl-3aa-hexahydroindane-4a-propionic acid, lB-carboxyl a-hydroxy-5-oxo-7aB-methyl-3aa-hexahydroindane-4a-propionic acid, IB-carboxymethyl-S-oxo- 7aB-methyl-3aa-hexahydroindane-4a-propionic acid, lB-carboxy-methyll a-hydroxy-S -oxo-7aB-methyl-3 aa-hexahydroindane-4a-propionic acid, lB-( l-carboxyethyl)-5-oxo- 7aB-methyl-3aB-hexahydroindane-4a-propionic acid, I ,B-( lcarboxyethyl l a-hydroxy-5-oxo-7aB-methyl-3aa-hexahydroindane-4a-propionic acid, 4a-(2-carboxyethyl)-5-oxo- 7aB, B-dimethyl-3aa-hexahydroindane-1 B-propionic acid, 401- (2-carboxyethyl)- l a-hydroxy-5-oxo-7aB, B-dimethyl-Saahexahydroindane-lB-propionic acid, 4a-( 2-carboxyethyl)-5- oxo-7a/3, 'y-dimethyl-3aa-hexahydroindane-lB-butyric acid, 4a-( 2-carboxyethyl l a-hydroxy-5-oxo-7afl, -y-dimethyl-3 aahexahydroindane-lB-butytic acid, 4a-(2-carboxyethyl)-5- oxo-7aB, 8-dimethyl-3aa-hexahydroindane-lB-valeric acid, 4a-( Z-carboxyethyl l -hydroxy-5 -oxo-7aB, 8-dimethyl-3aahexahydroindane-l fl-valeric acid, 4a-(2-carboxyethyl)-5-oxo- 7aB. e-dimethyl 3aa-hexahydroindane-lB-caproic acid, 401-(2- carboxyethyl)-l a -hydroxy-5 -oxo-7a,8, e-dimethyl-B aa-hex- 7a,8, as-trimethyl-3aa-hexahydroindane-lfi-caproic acid and their alkali metal salts.

It is to be noted that during the course of the reaction, the hydroxy group or alkanoyloxy group located at position 7 or 12 of the starting steroid compound disappears to give the corresponding 7- and 2-methylene group of the- 4-(2-carboxyethyl)-hexahydroindane skeleton.

The aforesaid products decrease plasma total cholesterol and phospholipid levels, and increase plasma total cholesterol/plasma phospholipid level ratio. For example, 40:- (2-carboxyethyl)-5-oxo-7a/3, y-dimethyl-3a-hexahydroindane-lB-butyric acid decreases plasma total cholesterol level l0.7 percent and phospholipid level l7.9 percent, and increases C/P ratio 7.9 percent in rats upon oral administration or subcutaneous injection, 1 mg X days, and l/8-( 1-carbox yethyl)-5-oxo-7aB-methyl-3aa-hexahydroindane-4apropionic acid decreases plasma total cholesterol level -8.0 percent and phospholipid level 7.5 percent and increases C/P ratio 3 percent in rats on oral administration or subcutaneous injection, 5 mg X 10 days. They can be administered to man or warm-blooded animals suffering from hypercholesterolemia or hypertension in the form of conventional preparations such as injections, tablets, powders, pills, granules, liquids, etc. A suitable daily dose is 0.1 100 mg/day per kg of body weight.

The following examples serve to illustrate practical embodiments of the present invention, but they are not intended to limit the scope of the invention. Percentages are by weight.

EXAMPLE 1 A culture medium 1.1 liters) of the following constitution:

Ammonium sulfate 0.2 Potassium dihydrogen phosphate 0.] Magnesium sulfate (heptahydrate) 0.05% Potassium chloride 0.05% Ferrous sulfate (heptahydrate) 0.01% Yeast extract 0.05% Sodium cholate 0. l%

is adjusted to pH 7.15 by addition of 10 percent-aqueous sodium hydroxide solution and divided into ml portions, which are severally placed in 500 ml Sakaguchis shaking bottles. After sterilization, the bottles are inoculated with Corynebacterium simplex (IFO 3530 or ATCC 6967 strain) and thereafter cultivated at 28 C. under shaking. After completion of the cultivation (about 4-5 days), the culture broth is adjusted to pH 4.0 by addition of 10 percent-aqueous hydrochloric acid solution and extracted with ether or ethyl acetate. The extract is washed with water, dried with anhydrous sodium sulfate and distilled to remove solvent. The crystalline residue (235 mg) afiords prismatic 4a-(2-carboxyethyl)-5-oxo-7afi, 'y-dimethyl-Elaa-hexahydroindanel B-butyric acid (M.P. l69.5-170 C, yield: about 20-30 percent). [11],, 20.5 i 5 (c 0.45, in ethanol). IR: v,,,,,,.' 3,l50-2,700, 1,703 cm". UV: no characteristic absorption band. Anal. Calcd. for C I-[ 0 C, 66.64; H, 8.70. Found: C, 66.71; H, 8.70.

EXAMPLE 2 Repitition of the treatment according to Example 1, except for utilization of ox bile extract (oxgall) in 0.1 0.2 percent concentration instead of 0.1 percent of sodium cholate, in the same culture medium, afiords the same product in a yield of about 20 percent.

EXAMPLE 3 Repetition of the treatment according to Example 1, except for replacement of sodium cholate by an equal amount of sodium lithocholate in the same culture medium, afi'ords the same product in a yield of about 20 percent.

EXAMPLE 4 Repetition of the treatment according to Example 1, except for replacement of sodium cholate by 0.1 percent of an equimolar mixture of sodium cholate and sodium chenodeoxycholate in the said cultivation medium, affords the same product in an about 20 percent yield.

EXAMPLE 5 Repetition of the treatment according to Example 1, except for replacement of sodium cholate by 0.1 percent of an equimolar mixture of sodium cholate and sodium deoxycholate in the same cultivation medium, affords the same product in an about 20 percent yield.

EXAMPLE 6 Essentially the same result is obtained by replacement of the culture medium in Example 1 by the following (pH 7. l 5-7.4):

' Ammonium sulfate 0.2 Dipotassium hydrogen phosphate 0.1 Magnesium sulfate (heptahydrate) 0.5% Ferric chloride (hexahydrate) 0.001% Sodium cholate 0.1

EXAMPLE 7 The addition of 0.05 percent of yeast extract to the culture medium in Example 6 gives the same result.

EXAMPLE 8 A culture medium (50 liters) of the same constitution as in Example 1, except for replacement of pure sodium cholate by 50 grams of crude crystalline cholic acid (prepared from ox bile extract, containing around 20 percent of desoxycholic acid), is inoculated with bouillon liquor (500 ml) precultured with Corynebacten'um simplex at 28 C. for 24 hours and aerobically cultured at 28 C. for 48 hours under reciprocal shaking at 375 r.p.m. The culture broth is adjusted to pH 2.0 and extracted with ethyl acetate. The extract is counter extracted with dilute aqueous sodium hydrogen carbonate solution. The alkaline aqueous extract is acidified with hydrochloric acid and then extracted with chloroform. The chloroform layer affords on evaporation the crude product, which is treated with acetone and gives 4a-(2-carboxyethyl)-5-oxo-7a[3, 'ydimethyl-3aa-hexahydroindane-1 B-butyric acid in about 30 percent yield. 1

The substance is dissolved in ethanol, added to an equimolar amount of sodium hydroxide and the mixture is concentrated, whereby the corresponding disodium salt (M.P. 300 C.) is obtained. It is soluble in water and reverts to the free acid form by the action of acid. By similar treatment, the corresponding dipotassium salt (M.P. 300 C.) is obtained.

EXAMPLE 9 The same treatment as in the Example 1 with the culture medium of the same constitution as in Example 1 except for replacement of sodium cholate by an equal amount of sodium 3-oxo-androst-4-ene-17B-carboxylate affords crude product in around 80 percent yield. By recrystallization from acetone, it is purified and affords lB-carboxy-5-oxo-7afimethyl-3aa-hexahydroindane-4a-propionic acid (M.P. 236.5-240.5 G). M1,, 34.5 1 2 (c. 1.064, chloroform). IR: v,,,,,,""- 3,l2,600, 1,694 cm. UV: no characteristic absorption band. Anal. Calcd. for C H O C, 62.67; H, 7.51. Found: C, 62.50; H, 7.50.

EXAMPLE l0 Corynebacterium simplex lFO 3530 strain) is precultured at 28 C, for 48 hours in a medium (1 liter) consisting of 0.5 per cent glucose, 0.5 percent polypeptone, 0.5 percent corn steep liquor and 0.1 percent sodium 3-oxo-androst-4-ene-l7B-carboxylate to make inoculant. A culture medium prepared in the same manner as in Example 1 by using 16.5 g of sodium 3-oxoandrost-4-ene-l7Bcarboxylate instead of sodium cholate is placed in a 30-liter jar fermenter, inoculated with the inoculant prepared above and then cultured at 30 C. for 114 hours under reciprocal shaking at 250-350 r.p.m., internal pressure 0.5 kg/cm and aeration rate 17 liters/min. The culture broth is adjusted to pH around 2 and extracted with ethyl acetate. The extract is washed with water and distilled to remove the solvent. The distillation residue is triturated with ether to give crude crystalline product (12.4 g), which afiords 9.06 g of pure crystals of 1fi-carboxy-S-oxo-7aB-methyl-3aa-hexahydroindane-4apropionic acid by recrystallization from methanol.

EXAMPLE 1 l The same procedure as Example 1 with replacement of the raw material sodium cholate by an equal amount of sodium 3B-hydroxy-23-norchol-5-en-22-oate affords crude lB-( l-car boxyethyl)--oxo-7aB-methyl-3aa-hexahydroindane-4apropionic acid in a yield of about 40 percent. On recrystallization from acetone-petroleum ether mixture, it gives pure crystals of M.P. l58.5 l59.5 C. [a],, 0.25" (c 3.620, chloroform), IR: V,,,,,,"' 3,200-2,800, 1,745, 1,710, 1,680 cm". UV: no characteristic absorption. Anal. Calcd. for C H- O. C, 68.84; H, 8.16. Found: C, 64.89; H, 8.13.

EXAMPLE l2 lnoculant is prepared by cultivation of 300 ml of medium of the same constitution as in Example 10 except for replacement of the sodium 3-oxo-androst-4-ene-l7B-carboxylate by sodium 3B-hydroxy-23-norchol-5-en-22-oate, at 28 C. for 72 hours. Culture media (2 X 20 liters) prepared in the same manner as in Example 10 by using sodium 3B-hydroxy-bisnorchol-S-enate (2 X 20 g) instead of sodium 3-oxo-androst-4- ene-l7ficarboxylate are placed in jar fermenters (2 X 30 liters), inoculated with the above inoculant and thereatter cultivated aerobically (aeration rate, 20 liters/min. at 29 32 C. for 45 hours under internal pressure of 0.5 kglcm at 250 r.p.m. The culture broth is adjusted to pH about land extracted with ethyl acetate. The extract is washed with water and distilled to remove solvent. The distillation residue is treated with ether and recovered 16.4 g of crude product, which affords crystalline pure product 1B-( l-carboxyethyl)-5 oxo-7aB-methyl-3aa-hexahydroindane-4a-propionic acid (8.61 g).

EXAMPLE 13 According to the procedure described in Example 12, 60 g of cholic acid (instead of 3B-hydroxy-23-norchol-5-en-22-oic acid) is cultivated in 3 X 30 liter-jar fermenters and treated, whereby 32.3 g of crude product 4a-( 2-carboxyethyl)-5-oxo- 7afl, 'y-dimethyl-Baa-hexahydroindane-lfi-butyric acid is obtained.

The same treatment with 3B-hydroxychol-5-enic acid affords the same product.

EXAMPLE 14 According to the procedure described in the above Example 12, but using a mixture consisting of sodium cholate and a compound selected from sodium 3-oxo-pregn-4-en-2l-oate, sodium 24-norcholate, sodium 3fi-hydroxy-27-nor-5B- cholestan- 26-oate and sodium 3-oxo-5a-cholestan-25-oate instead of sodium 3B-hydroxy-23-norchol-5-en-22-oate, the respective products, 1fl-carboxymethyl-S-oxo-7aB-methyl-3 aa-hexahydroindane-4a-propionic acid, 4a-( 2-carboxyethyl)- 5-oxo-7afi, B-dimethyl-Saar-hexahydroindane- 1 fl-propionic acid, 4a-(2-carboxyethyl)5-oxo-7afl, 8-dimethyl-3aa-hexahydroindane-lB-valeric acid, 4a-(2-carboxyethyl)-5-oxo- 7afi, e-dimethyl-3aa-hexahydroindane-IB-caproic acid and 4a-(2-carboxyethyl)-5-oxo-7a/:l, a, e-trimethyl-3aa-hexahydroindane-lB-caproic acid are obtainable as crystalline products.

EXAMPLE l5 lnoculant (700 ml X 4 is prepared from an aqueous solution containing 0.5 percent glucose, 0.5 percent polypeptone, 0.5 percent corn steep liquor, 0.1 percent sodium l7a-hydroxy-3-oxo-androst-4-ene-l7B-carboxylate by seeding Carynebacterium (Arthrobacter) simplex, followed by cultivation at 28 C. for 48 hours. Four jar-fermenters containing a total 73 liters of culture medium containing the following:

Ammonium sulfate 0.2 Potassium dihydrogen phosphate 0.1 Magnesium sulfate (heptahydrate) 0.05% Potassium chloride 0.05% Ferrous sulfate (heptahydrate) 0.01% Yeast extract 0.05% Sodium 17a-hydroxy-3-oxo-androst -4-ene-l7B-carboxylate 0.1

is sterilized by heating at C. for 20 minutes and adjusted with aqueous sodium hydroxide to pH 7.2. The inoculant prepared above is added into each fermenter. Cultivation is carried out under reciprocal shaking 300 r.p.m., internal pressure 0.5 kg/cm", aeration rate 23 liters/min, for 42 hours at 29 C. (the formation of the product is traced by thin-layer chromatograrns of a part of the culture broth). The culture broth is concentrated to one-eighth of the original volume under reduced pressure, aqueous hydrochloric acid added to adjust the pH to 2.0 and extracted with ethyl acetate. The organic layer is washed with water, dried and concentrated. The separated crystals are collected by filtration, and ether is added to the mother liquor and the separated crystals are collected by filtration and combined with the crystals obtained above. Forty-four grams of crude crystals are recrystallized from acetone-ether to give 43.1 grams of lfl-carboxy-lahydroxy-S-oxo-7a,B-methyl-3aa-hexahydroindane-4apropionic acid, M.P. 151 153 C. Yield 66.3 percent of pure product. Analytical sample melts at 153 154 C. [01],, 32 2.6 0.4 (c 0.962, ethanol). 1R: v f' 3,494, 3,450, broad peak at around 2,666, 1,698 cm". Anal. Calcd. forC,.,l-l, O C, 59.14; H, 7.09. Found: C, 59.37; H, 7.15.

EXAMPLE 16 According to the procedure described in the above Example l, but using sodium 3-oxo-l7a-hydroxypregn-4-ene-2O- carboxy late instead of sodium cholate, there is obtained 4a- (2-carboxyethy1)-1a-hydroxy-5-oxo-7al3,

hexahydroindane-1/3-acetic acid.

EXAMPLE 17 a-dimethyl-3 aasalts or dipotassium salts, having the respective melting points higher than 300 C.

What is claimed is:

l. A member selected from the group consisting of compounds of the formula:

and the alkali metal salts thereof, wherein X means the absence or presence of a grouping selected from the group consisting of CH CH(CH and Cl-l(Cl-l )-(CH in which n is an integer 1 to 4 inclusive.

.2. A compound according to claim 1, namely lB-carboxy-l a-hydroxy-S-oxo-7aB-methyl-3aa-hexahydroindane-4apropionic acid.

3. A compound according to claim 1, namely 1B-( l-carboxyethyl)- 1a-hydroxy-S-oxo-7aB-methyl-Saa-hexahydroindane- 4a-propionic acid.

t i t I v UNITED STATES PATENT OFFICE CERTIFICATE OF CORECTIUN Patent No. 3.674.842 Dated Julv 4. 1972 Inventor) Shoh'ei Hayakawa, Yoshiko Kanomatau and 'lfakuel-ll l uj iwam It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Page one, first; column, below the line reading [21] Appl., No, 2

75,728", insert the following:

-- [30] Foreign Application Priority Data August 14, 1964 Japan; 46852/64' si ned and'sealed this 8th day of October 19740 (SEAL) Attest:

MCCOY M. GIBSON JR. 0. MARSHALL 'DANN Attesting Officer Commissioner of Patents uscoMM-oc scan-Poo A U.S. GOVERNMENT PRINTING OFFICE 2 I959 O-396'35,

F ORM PO-105O (10-69) 

2. A compound according to claim 1, namely 1 Beta -carboxy-1 Alpha -hydroxy-5-oxo-7a Beta -methyl-3a Alpha -hexahydroindane-4 Alpha -propionic acid.
 3. A compound according to claim 1, namely 1 Beta -(1-carboxyethyl)-1 Alpha -hydroxy-5-oxo-7a Beta -methyl-3a Alpha -hexahydroindane-4 Alpha -propionic acid. 